cistus laurifolius

457
The methodology described by Kurtel et al. (1992) was
used. Briefly, 1 ml of plasma was mixed with 2.0 ml of
trichloroacetic acid (TCA; 15%, w/v)–thiobarbituric acid (TBA;
0.375%)–0.25 N HCl and mixed throughly and centrifuged at
10,000 for 5 min. The supernatant was mixed with 20 l
of butyl hydroxy toluene (BHT; 0.02% in 95% EtOH, w/v) to
prevent further oxidation and heated for 15 min in a boiling
water bath. After cooling under running water, the flocculent
precipitate was removed by centrifugation at 10,000 for
5 min. The absorbance of the sample was measured at 532 nm
against blank that contained all the reagents except plasma.
1,1,3,3-Tetraethoxypropan was used as standard for the curve
calibration.
with no homogenate. Results were expressed as
tissue.
mol GSH/g
Biocon standard kits and DAX-48 autoanalyzer were used to
measure AST and ALT activities in plasma samples according
to the method of Wilkinson et al. (1972).
Animals employed in the experiments were observed during
48 h and morbidity or mortality was recorded, if happens, for
each group at the end of observation period.
The method of Ohkawa et al. (1979) as modified by Jamall
and Smith (1985) was used to determine lipid peroxidation in tissue
samples. Mice were sacrified by an overdose of diethylether.
The liver of each mouse was immediately excised and chilled in
ice-cold 0.9% NaCl and then perfused via the portal vein with
ice-cold 0.9% NaCl. After washing with 0.9% NaCl, 1.0 g of wet
tissue was weighted exactly and homogenized in 9 ml of 0.25 M
sucrose using a teflon homogenizer to obtain a 10% suspension.
The cytosolic fraction was obtained by a two-step centrifugation
first at 1000 for 10 min and then at 2000 for 30 min
at 4 C. A volume of the homogenate (0.20 ml) was transferred
to a vial and was mixed with 0.2 ml of a 8.1% (w/v) sodium
dodecyl sulphate solution, 1.50 ml of a 20% acetic acid solution
(adjusted to pH 3.5 with NaOH) and 1.50 ml of a 0.8% (w/v)
solution of TBA and the final volume was adjusted to 4.0 ml
with distilled water. Each vial was tightly capped and heated in
boiling water bath for 60 min. The vials were then cooled under
running water.
Equal volumes of tissue blank or test sample and 10%
TCA were transferred into a centrifuge tube and centrifuged at
1000 for 10 min. The absorbance of the supernatant fraction
was measured at 532 nm in a Beckman DU 650 spectrometer.
Control experiment was processed using the same experimental
procedure except the TBA solution was replaced with distilled
water due to the peroxidative effect of acetaminophen on tissue.
Livers of acetaminophen-treated mice were used as positive control
and 1,1,3,3-tetraethoxypropan was used as standard for the
curve calibration.
Some 200 mg of liver was homogenized in 8.0 ml of 0.02 M
EDTA in an ice bath. The homogenates were kept in the ice bath
until used. Aliquots of 5.0 ml of the homogenates were mixed
in 15.0 ml test tubes with 4.0 ml distilled water and 1.0 ml of
50% trichloroacetic acid (TCA). The tubes were centrifuged for
15 min at approximately 3000 . Some 2.0 ml of supernatant
was mixed with 4.0 ml of 0.4 M Tris–buffer, pH 8.9, 0.1 ml Ellman’s
reagent [5,5 -dithiobis-(2-nitro-benzoic acid)] (DTNB)
added, and the sample shaken. The absorbance was read within
5 min of the addition of DTNB at 412 nm against a reagent blank
The data obtained were analyzed by one-way of variance
(ANOVA) and Student–Newman–Keuls post hoc tests for the
significant interrelation between the various groups using Instat
computer software. < 0.05 was considered to be significant
different from the control.
In living systems, dietary antioxidants such as -tocopherol,
ascorbic acid, carotenoids, as well as flavonoids and related
phenolic compounds are suggested in protection from oxidative
damage of tissues in the body and eventually for a healthy
life (Haraguchi, 2001). Especially flavonoids have been shown
to scavenge various reactive oxygen species and implicated as
inhibitors of lipid peroxidation (Mora et al., 1990).
Acetaminophen (paracetamol), a frequently used analgesic
and antipyretic drug, is known to be hepatotoxic in high doses,
which is primarily metabolized by sulfation and glucuronidation
to unreactive metabolites, and then activated by the cytochrome
P-450 system to produce liver injury. It is established that
acetaminophen is bioactivated to a toxic electrophile, -acetyl-
-benzoquinone imine (NAPQI), which binds covalently to tissue
macromolecules, and probably also oxidizes lipids, or the
critical sulphydryl groups (protein thiols) and alters the homeostasis
of calcium (Lin et al., 1997). Post-mitochondrial supernatants
isolated from livers of rats given a single large oral dose
of acetaminophen (800 mg/kg) showed rapid rates of lipid peroxidation
when incubated in vitro. Lipid peroxidation probably
occurs simultaneously with the proposed covalent binding of
the active metabolite of acetaminophen. Since the former process
is known to cause severe and extensive membrane damage,
it may be a very important factor in acetaminophen-induced liver
necrosis (Fairhurst et al., 1982).
As shown in Table 1, in the liver and plasma of
acetaminophen-treated group, tissue and plasma lipid peroxidation
levels (219.2 and 1172%) as evidenced by MDA determination
increased significantly as compared to control group, however,
the content of GSH in liver decreased (8.7%). Additionally,
acetaminophen was found to cause several folds increases in
plasma AST and ALT levels (106 and 71.6%). Ethanol (EtOH)
extract of
leaves administered in 500 mg/kg